A current major focus of the laboratory is investigation of the physiological functions of the neuropeptide Tuberoinfundibular Peptide of 39 residues (TIP39) and its receptor the Parathyroid Hormone 2 (PTH2) receptor, which were discovered in this laboratory several years ago. TIP39 is synthesized by two discrete groups of neurons that project to areas involved in emotional function. These areas contain a matching distribution of the PTH2 receptor, and neurons in these regions project to the areas containing TIP39 neurons. Thus the system is ideally positioned to coordinate and modulate functions relevant to mental disorders. Our experimental work is performed in rodents, but to evaluate its relevance to human behavior we compared the neuroanatomical distribution of TIP39 and PTH2 receptor expression in humans and non-human primates with the distribution in rodents. During this reporting period we completed a study of the neuroanatomical distribution of the PTH2 receptor and TIP39 that showed that they are extremely similar to each other in human and non-human primates, and to the distributions that we have previously reported in rat and mouse. This suggests that it should be possible to extrapolate from what we learn about this system in rodents to humans. In primates as well as rodents TIP39 containing fibers and PTH2 receptor synthesizing neurons are concentrated in brain regions involved in emotion and hormonal regulation. There is a high TIP39/PTH2 receptor density in the area of the hypothalamic paraventricular nucleus, which is a major regulator of pituitary hormone release. A high degree of colocalization between the PTH2 receptor and a vesicular glutamate transporter indicates that PTH2 receptors are present on glutamatergic neurons. PTH2 receptor containing glutamatergic nerve terminals are adjacent to cells containing each of the major pituitary regulating hormones in this region, suggesting that TIP39 may regulate pituitary function by modulating release of the neurotransmitter glutamate. We tested this idea by infusing TIP39 into the hypothalamic paraventricular nucleus in the presence and absence of drugs that block ionotropic glutamate receptors. We found that TIP39 activated a large number of hypothalamic neurons and that this activation was dependent on the activation of ionotropic glutamate receptors. The infusion of TIP39 also affected the levels of several hormones in the bloodstream. Thus TIP39 appears to be an endogenous regulator of neuroendocrine function, acting by modulating the excitation of cells containing hypothalamic hormone releasing factors. Previously, we found using established behavioral tests such as the open-field, dark-light emergence, shock-probe burying and elevated plus maze under varied lighting or following restraint, that mice with null mutation of the gene encoding TIP39 (TIP39-KO) have increased anxiety-like behavior when stressed. We also found that they exhibit greater fear-like behavior in a Pavlovian conditioning paradigm. The increased fear-like behavior (freezing behavior in the test) appeared to result from increased learning or increased significance of cues and not to a change in extinction of a learned association. We are continuing our investigation of the phenotype of TIP39-KO mice and have developed mice with a null mutation and mice with a conditional null mutation of the gene encoding the PTH2 receptor. We have begun using a more ethologically relevant novel object recognition paradigm to investigate the role of TIP39 signaling. Current evidence supports the suggestion that TIP39 signaling normally modulates behavioral responses to stress. TIP39 and the PTH2 receptor are also present in several brain areas that are part of pain processing pathways. Previous data, using acute administration of synthetic TIP39, supported the suggestion that TIP39/PTH2 receptor signaling modulates pain information. Surprisingly, we found little difference in pain behavior assays in knockout mice. We recently developed a PTH2 receptor antagonist. We used this antagonist to reexamine the potential involvement of TIP39 signaling in pain. Using intrathecal and intracerebroventricular administration we find robust effects in nociceptive assays, and comparing the effects of the antagonist in wild-type and knockout mice clearly shows that the effects are due to block of TIP39 signaling. Greater potency of the antagonist following intracerebral infusion than intrathecal administration, and TIP39 and PTH2 receptor expression in brain areas involved in affective aspects of pain processing such as midline thalamic and amygdalar nuclei suggest that TIP39 signaling is involved in emotional aspects of pain processing. Our interpretation of these data is that TIP39 may normally function to modulate several mood-related functions. We speculate that dysregulation of TIP39 or genes like TIP39 could increase the vulnerability to psychiatric disorders and that endogenous regulation of TIP39 signaling may limit dysfunctional responses that could lead to mood or anxiety-related disease. Chronic pain, anxiety, and depressed mood individually present major public health problems and they also contribute to the burden of many other diseases. Increased understanding of the neuromodulatory systems that affect these symptoms will ultimately lead to better intervention strategies.